Mitochondria are multi-faceted organelles in eukaryotic cells that stand at the nexus of energy metabolism, oxidative stress, and apoptosis. Consequently, circumstances (genetics, environmental factors, aging) that result in mitochondrial dysfunction disrupt a multitude of cellular processes that can cause human disease pathology, ranging from heart, skeletal muscle and nerve dysfunction to diabetes, blindness, and deafness. Of course, a major function of mitochondria is to generate ATP through the process of oxidative phosphorylation (OXPHOS). ATP is the main energy currency in cells and tissues that require sustained energy production to perform their physiological function (e.g. muscle, nerves, and major organs such as heart and brain). Mitochondria contain a DNA genome (mtDNA), which in humans encodes thirteen essential protein components of the mitochondrial OXPHOS complexes and is maternally inherited. Knowledge of the unique mechanism of human mtDNA expression is essential to understand how this important cellular organelle functions and contributes to human diseases and age-related pathology. While defining the core factors and mechanisms required has advanced significantly through the efforts of my lab and others, only a handful of mitochondrial factors involved in events downstream of transcription initiation in human mitochondria have been characterized to date and their mechanisms of action are largely unknown. These significant gaps in our knowledge of human mitochondrial gene expression are addressed in this competitive renewal proposal through the following three specific aims: 1) To determine how altering mitochondrial gene expression affects metabolism and provides resistance to diet-induced obesity using the Tfam+/- mouse model, 2) To determine the mechanism of transcriptional activation of free mitochondrial ribosomal protein L12, and 3) To dissect the multiple functions of the Leigh Syndrome protein LRP130 in transcription and post-transcriptional processes in human mitochondria. Altogether the proposed studies are part of a larger continuum and strategy over the last 15 years on this project in my lab, which is to 1) identify new factors involved in human mitochondrial gene regulation, 2) determine their mechanism of action, and 3) to study how they influence cellular physiology and metabolism and contribute to human disease using mouse models. In the current proposal, Aim I is at stage 3 of this process (in vivo studies in mice/disease relevance), while Aims II and III are at stage 2 (determining mechanisms of action of new factors), but also employ a novel strategy for identifying new key players in this important process (i.e. stage 1). Knowledge gained from these studies holds therapeutic promise for mitochondrial disease and age-related pathology based on augmenting mitochondrial gene expression and directly addresses the human disorders obesity and Leigh Syndrome.